Crayfish shells-derived carbon dots as a fluorescence sensor for the selective detection of 4-nitrophenol

ABSTRACT

As a type of environmentally deleterious compound, 4-nitrophenol (4-NP) has become a global concern due to its extreme toxicity to aquatic organisms and humans. Herein, we used naturally available crayfish shells as precursors to prepare a new type of carbon dots (CDs) by the hydrothermal method. The as-synthesized CDs exhibited strong blue fluorescence emission which can be sensitively quenched by 4-NP through the static quenching effect because of the formation of a ground state complex between CDs and 4-NP. Accordingly, a facile and rapid fluorescence sensor was developed to detect 4-NP from 0 μM to 50 μM with a limit of detection (LOD) of 0.16 μM. Furthermore, the proposed sensor was successfully applied for the detection of 4-NP in fresh crayfish meat and aquatic water samples.

KEYWORDS:

• Crayfish shells
• carbon dots
• fluorescence sensors
• 4-nitrophenol
• quenching mechanism

Introduction

With the development of the industry, environmental and food safety issues caused by pollutants such as heavy metals (Zaynab et al., 2022), pesticide residues (Wang et al., 2021), antibiotics (Gothwal & Shashidhar, 2014), and phenolic compounds (Patel et al., 2020) have attracted more and more attention. 4-nitrophenol (4-NP), as a common and important phenolic compound, has been frequently utilized in the chemical, pharmaceutical and military industries, such as the fabrication of various pesticides, dyes, explosives and pharmaceuticals (Han et al., 2019; Li et al., 2020). 4-NP can pose extremely deleterious effects on humans such as damage to the liver, kidneys and central nervous system through oral, skin or respiratory tract (Yin et al., 2016). Accordingly, it is among the list of priority pollutants and its maximum permissible concentration in drinking water has been established by the US Environmental Protection Agency (EPA) as 0.43 μM (Zhu et al., 2021). Furthermore, due to the poor biodegradability and high stability of 4-NP, it can accumulate in organisms through the food chain, thus posing a serious threat to fishery products’ safety and human health. Therefore, it is necessary to establish a rapid, convenient and effective method for sensitive monitoring of 4-NP in aquatic water and food samples.

Currently, a number of traditional methods have been developed for 4-NP detection, including spectrophotometry (Xia et al., 2021), high-performance liquid chromatography (HPLC) (Faraji et al., 2020), capillary electrophoresis (Fischer et al., 2006) and electrochemical methods (Zhang et al., 2017). Unfortunately, these methods suffer from some disadvantages such as expensive and sophisticated instruments, cumbersome pre-processing procedures, low selectivity and time-consuming detection processes.

Lately, carbon dots (CDs), as a new class of zero-dimensional carbon nanomaterials, have been extensively used as fluorescent probes in the field of contaminant analysis due to tunable photoluminescence (PL), excellent solubility, low toxicity, chemical inertness and good biocompatibility (Gu et al., 2021; He et al., 2015). To date, scientists have been devoting increasing efforts to the synthesis of CDs from various carbon sources (lemon juice, bamboo leaf, algae, the flowers of Abelmoschus manihot (Linn.) etc.), as PL behaviours of CDs depend on the precursor material (Kaur et al., 2017; Singh et al., 2020; Wan et al., 2019; Yang et al., 2020). However, the latest trend in CD synthesis is to use low-value and abundant man-made wastes as raw materials to reduce preparation costs, save resources, and minimize environmental impacts (Hu & Gao, 2020; Wang et al., 2020b). In addition, a more feasible and desirable option is to utilize various heteroatoms (such as N, S, and P) in waste resources to dope CDs, to effectively improve the quantum yield and provide more modifiable groups (Liu et al., 2019).

Crayfish are well received by consumers around the world because of their delicious taste and abundant nutrition. Currently, China is the world's largest crayfish producer and consumer, producing more than 2.4 million tons during the year 2020 (Tan et al., 2021). Meanwhile, a large amount of crustacean waste is generated in the production of crayfish products, which can pose a serious environmental problem if discarded they are difficult to biodegrade, insoluble in water, and expensive to dispose of. Crayfish consist mainly of chitin, calcium-type minerals, protein and chitin can be employed as excellent precursors rich in carbon and nitrogen to fabricate nitrogen-rich CDs (Wang et al., 2022).

Herein, we presented a cost-effective and sustainable route for the green synthesis of CDs from low-value waste crayfish shells by the hydrothermal method. The as-prepared CDs exhibited bright blue light with a quantum yield of 10.68% and showed a specific fluorescence quenching towards 4-NP, which rendered them an ideal fluorescent probe for 4-NP sensing. The proposed method can detect 4-NP selectively and sensitively in the range of 0–50 μM, with a LOD of 0.16 μM. According to the investigation, the fluorescence quenching mechanism was attributed to static quenching. Furthermore, the analytical performance of this method was demonstrated in fresh crayfish meat and aquatic water samples with a recovery range of 95.80–103.55%.

Materials and methods

Reagents

The analytical reagents, such as Hydrogen chloride (HCl), Sodium hydroxide (NaOH), KCl, NaCl, CaCl₂, MgSO₄, MnSO₄, BaCl₂, Zn(Ac)₂, FeCl₂, CuCl₂, FeCl₃ and the test anions, were supplied by the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Phenol, p-dihydroxybenzene (p-DNB), o-dihydroxybenzene (o-DHB), m-dihydroxybenzene (m-DHB), 2-nitrophenol (2-NP), 3-nitrophenol (3-NP), 4-nitrophenol (4-NP), p-cresol (p-MP), p-phenylenediamine (p-PD), p-chlorophenol (p-CP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) were provided by Aladdin Chemical Co., Ltd. (Shanghai, China). All the chemicals were used at the least level of analysis. All aqueous solutions were prepared in ultrapure water. All other chemicals were of analytical grade and used as received. Ultrapure water (18.2 MΩ cm) was utilized throughout the experiments.

Instrumentation

The ultraviolet–visible (UV-vis) absorption spectra were measured using UV-vis UV-1800 spectrophotometers (Shimadzu Ltd., Japan). The excitation and emission of fluorescence spectra were performed on a fluorescence RF-6000 spectrophotometer (Shimadzu Ltd., Japan). The transmission electron microscopy (TEM) images of CDs were obtained from a JEM-2100 apparatus (JEOL Ltd., Japan). The diameter of CDs was determined by a ZS-90 Zetasizer Nano (Malvern Instruments Ltd., UK). The Fourier-transform infrared (FTIR) spectrum was obtained using a Nicolet 6700 FTIR spectrometer (Nicolet Ltd., USA). The X-ray diffraction (XRD) study of CDs was carried out with a Bruker D8 Advance X-ray diffractometer (Bruker Instruments Ltd., Germany). The X-ray photoelectron spectroscopy (XPS) spectra were performed on an Escalab 250Xi XPS (Thermo Scientific, USA).

Pretreatment of crayfish shells

The crayfish were bought from the local market (Wuxi, China). They were shelled and the shells were rinsed under running water and then washed with deionized water. The cleaned crayfish shells were dried at 70°C for 12 h. The dried shells were pulverized and sifted via an 80-mesh sieve. The dried sample was treated with 0.4 M HCl (1:20 w/v) for 3 h with ultrasonic assistance and then washed with ultrapure water to neutral pH. After that, the sample was transferred into 1 M NaOH (1:20 w/v) at 80°C and treated for 6 h. The obtained product was then washed with ultrapure water to neutral pH and dried.

Synthesis of CDs

The obtained crayfish shells sample (1 g) was dispersed in 25 mL ultrapure water and ultrasound for 10 min. Then the mixture was transferred to a 45 mL Teflon-lined stainless-steel autoclave and kept at 180°C for 8 h. After that, the obtained sample was centrifuged under 11,000 rpm for 15 min. The collected supernatant was filtered through a 0.22 μM hydrophilic membrane and further dialyzed in a cellulose membrane cut to 1000 Da molecular weight for 48 h. Finally, the purified brown colour solution was stored at 4°C for further characterization and application.

Sensing of 4-NP

The assay of 4-NP was carried out at room temperature in a PBS (0.2 M, pH 6.0) solution. In a typical experiment, 200 mg of CDs was added into 1000 mL of a PBS solution to form a 0.2 mg mL⁻¹ dispersion. Then, 100 μL of 4-NP solution with different concentrations (0.5–100 μM) was added into 200 μL of the above CD dispersion. The mixture was shaken thoroughly for 1 min. After that, the fluorescence spectra with a recording emission range of 380–650 nm were collected at the excitation wavelength of 350 nm. In the selectivity experiments, 4-NP was replaced by common ions including K⁺, Na⁺, Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Fe³⁺, F^-, Cl^-, NO₃^-, CO₃^2-, SO₄^2-, and some several structurally similar competitive compounds such as phenol, p-DNB, o-DHB, m-DHB, 2-NP, 3-NP, 4-NP, p-MP, p-PD, p-CP, 2,4,6-TCP, PCP, and the detection method was the same as 4-NP analysis.

Determination of 4-NP in real samples

Water samples were collected from aquatic water in Jiangsu. First, water samples were purified through centrifugation at 10,000 rpm for 10 min and filtered by a 0.22 μm hydrophilic membrane filtration. After that, the obtained water samples were mixed with the standard 4-NP solution (0, 5, 10, and 20 μM) to prepare spiked samples. Different concentrations of 4-NP (0, 5, 10, and 20 μM) were added to fresh crayfish meat samples (2 g) and mixed overnight. The mixtures were crushed thoroughly with a homogenizer and dispersed into PBS (0.2 M, pH 6.0) solution. The samples were vortex mixed for 1 min, ultrasonic extraction for 5 min, and then centrifuged to collect the supernatant. After centrifugation, the residue was repeatedly treated with PBS (0.2 M, pH 6.0) solution, and the supernatant was combined and mixed. Finally, the levels of 4-NP in water samples and crayfish meat samples were determined through the sensor mentioned above. The data of 4-NP fluorescence spectra were then compared with that were measured using a traditional method like high-performance liquid chromatography (HPLC–UV) (Xu et al., 2021).

Results and discussion

Synthesis and optical properties of CDs

In this study, naturally derived crayfish shells were employed as raw material to prepare CDs via a single-step hydrothermal method. During the preparation processes, three main factors (including reaction temperature, reaction time, and solid–liquid ratio) affecting the fluorescence QY of the CDs, were optimized at first. It is clear from Table S1 that the CDs with the highest QY of 10.68% were prepared by using 1 g of crayfish shells as raw materials, 25 mL ultrapure water as the solvent, 180°C as reaction temperature, and 8 h as reaction time.

The optical properties of CDs were investigated by UV-vis absorption spectrum and fluorescence spectrum. The inset in Figure 1(A) shows that the as-obtained CDs were almost colourless in visible light and exhibited bright blue fluorescence under a 350 nm ultraviolet lamp. Additionally, from Figure 1(A), the UV-vis absorption spectrum indicated that there was an absorption peak located at 273 nm, corresponding to π-π* electronic transition of C = C (Gu et al., 2021). The red line and black line in Figure 1(A) were associated with the optimal excitation wavelength and the maximum emission wavelength at 350 and 430 nm, respectively. As shown in Figure 1(B), a slight redshift in the fluorescence emission peak from 400 to 482 nm was observed when the excitation wavelength increased from 300 nm to 400 nm. This behaviour was consistent with what has been reported previously that the emission wavelength is dependent on the excitation wavelength. This phenomenon was attributed to the different size distribution and various surface functional groups of CDs (Wan et al., 2019).

Figure 1. (A) UV-vis absorption, fluorescence excitation and emission spectra of CDs (the inset pattern is photographs of CDs under visible light and UV light). (B) Fluorescence emission spectra of CDs at different excitation wavelengths.

In order to further study the stability of CDs, the effects of pH value, ionic strength and UV radiation time were investigated, and the results are shown in Figure S1A. The fluorescence intensity of CDs remained invariant when the pH was in the range of 5–10, but decreased obviously when the pH became acidic or alkaline, resulting from the protonation/deprotonation of surface groups or the change of surface charge density (Yang et al., 2020). In addition, the CDs’ fluorescence intensity was almost constant when the ionic strength increased from 0 to 5 M, which suggested that ionic strength had no significant effect on the CDs’ fluorescence (Figure S1B), as shown in Figure S1C. The fluorescence intensity of CDs had no significant difference under continuous irradiation for 12 h with a UV lamp, indicating that the CDs possessed outstanding photostability.

Characterization of CDs

The spherical morphologies of CDs were revealed using TEM images, as depicted in Figure 2(A). The prepared CDs exhibited spherical morphology and were well dispersed. The diameter of resulting CDs ranged from 2 to 5.5 nm with an average size of 3.8 nm (Figure 2(B). The XRD analysis was used to investigate the crystalline nature of the CDs. It can be seen from Figure 2(C) that the XRD pattern of CDs exhibited a wide diffraction peak at 2θ = 20.8°, suggesting the monodispersed and crystallized structure of the synthesized CDs (Kaur et al., 2017).

Figure 2. (A) TEM image, (B) corresponding size distribution, (C) XRD pattern, (D) FTIR spectrum of the CDs.

The functional groups of the prepared CDs were analyzed by FTIR spectrum, as shown in Figure 2(D). Broad peaks within the range of 3400–3000 cm⁻¹ were the stretching vibration of O-H/N-H, demonstrating amino and hydroxyl groups (Ding et al., 2021). The band observed at 1660 and 1072 cm⁻¹ may be designated as the C = O and C–O stretching vibration (Krishnaiah et al., 2022). Additionally, the sharp band at 1570 cm⁻¹ belonged to the stretching vibrations from the C = C/C = N functional groups (Liang et al., 2021). The characteristic vibrational absorption at 1400 cm⁻¹ was caused by C–N bond, which confirmed the doping of nitrogen (Sun et al., 2022).

The XPS analysis was performed to characterize the chemical structure and composition of CDs. As exhibited in Figure 3(A), the XPS full-scan spectrum showed three typical peaks at 284.8, 398.8 and 531.8 eV, which were related to C1s, N1s and O1s, respectively. The CDs contained 63, 7 and 30 atomic percentages of carbon, nitrogen and oxygen, respectively. In the high-resolution XPS spectra (Figure 3(B–D)), The C1s spectrum showed three major component bands at 284.8, 285.4, 287.1 and 287.8 eV, corresponding to C–C, C–N, C–O and C = N, respectively (Han et al., 2019). The N1s spectrum proved the existence of pyridinic N (398.6 eV), pyrrolic N (399.3 eV) and graphitic N (400.5 eV) (Gu et al., 2021). The O1s spectrum can be deconvoluted into three prominent bands at 530.8, 531.7 and 532.8 eV, which are attributed to C = O, C–O-H and C–O-C, respectively (Gu et al., 2021; Qi et al., 2019). The results of the XPS analysis were consistent with those of the FTIR spectrum, further confirming that the surface functional groups of CDs contained -OH, -NH₂, and -COOH, which are favourable for the sensing applications of CDs.

Figure 3. (A) XPS survey spectrum of the prepared CDs. High-resolution XPS spectra of C1s (B), N1s (C), and O1s (D).

Fluorescence assay of 4-NP

To achieve superior analytical performance for the sensing of 4-NP, the experimental conditions (including the concentration of CDs, pH, and the reaction time) were optimized. Respective results are shown in Figure S2. The fluorescence intensity of the detection system at 430 nm was measured and calculated by adding 4-NP with various concentrations into the CD solution under the above optimal conditions. Figure 4(A) exhibits that the fluorescence intensity of CDs decreased gradually with the increase of 4-NP concentration that ranged from 0 to 100 μM. Figure 4(B) shows the relationship between the quenching ratio F/F₀ and 4-NP concentration, and a good linear correlation can be calculated as F/F₀ = −0.0156 C_4-NP + 1.002 with a coefficient of determination (R²) of 0.996. The LOD was determined to be 0.16 μM based on a signal-to-noise ratio of 3, which was lower than the maximum allowable level (0.43 μM) in drinking water guided by USEPA.

Figure 4. (A) Fluorescence response of CDs with different 4-NP concentrations (0–100 μM) (the inset pattern is photographs of CDs under 350 nm excitation in the presence of 4-NP at concentrations of 0, 10, 30, 50 and 100 μM, respectively). (B) The relationship between F/F₀ and 4-NP concentration from 0 to 50 µM. Selectivity of CDs for 4-NP in the presence of various common inorganic ions (C) or structurally similar competitive compounds (D).

Subsequently, in order to further assess the selectivity of the CDs sensor towards 4-NP, a variety of common inorganic ions, such as K⁺, Na⁺, Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Fe³⁺, F^-, Cl^-, NO₃^-, CO₃^2- and SO₄^2-, were tested as a contrast. It was evident from Figure 4(C), these common inorganic ions did not induce remarkable fluorescence change on the detection of 4-NP. Moreover, several structurally similar competitive compounds, including phenol, p-DNB, o-DHB, m-DHB, 2-NP, 3-NP, 4-NP, p-MP, p-PD, p-CP, 2,4,6-TCP and PCP, were also used to evaluate the selectivity. As depicted in Figure 4(D), these compounds caused negligible interference to the fluorescence of the detection system except for the weak effect of 2-NP on fluorescence quenching. The analysis further proved that this method had excellent selectivity for 4-NP and can be further applied to the detection of 4-NP in real samples.

Possible fluorescence quenching mechanism

It is well known that fluorescence quenching may be induced by the static quenching effect (SQE), the dynamic quenching effect (DQE), Förster resonance energy transfer (FRET), the inner filter effect (IFE), photoinduced electron transfer (PET) (Hu et al., 2022). In this study, the fluorescence of CDs was obviously quenched by 4-NP, thus a series of experiments were performed to illuminate the exact quenching mechanism. It can be seen from Figure 5(A) that the UV-vis absorption spectrum of CDs changed significantly in the absence and presence of 4-NP, resulting from the formation of a complex between CDs and 4-NP. As shown in Figure 5(B), there was an evident overlap between the absorption spectrum of 4-NP and the excitation spectrum of CDs, suggesting that 4-NP can effectively shield the excitation light given to CDs, which is a notable feature of static quenching (Wang et al., 2020a). To further explore the mechanism of fluorescence quenching, the experiments were carried out at different temperatures to evaluate the effect of temperature on the stability of the ground state complex. Fluorescence quenching efficiency at different reaction temperatures was measured as displayed in Figure 5(C) and Table S2, and the K_s values decreased when the reaction temperature increased. The increase in temperature will lead to a decrease in the stability of the ground state complex, thus reducing the effect of static quenching (Zu et al., 2017). This further demonstrated that the fluorescence quenching between CDs and 4-NP is a static quenching process.

Figure 5. (A) The UV-vis absorption spectra of CDs, 4-NP, and the mixture of CDs and 4-NP. (B) The UV-vis absorption spectrum of 4-NP, fluorescence excitation, and emission spectra of CDs. (C) Stern-Volmer values of the CDs/4-NP at different temperatures.

Real sample analysis

To evaluate the actual performance of our proposed method, a traditional HPLC-UV method and our CD system were both performed in real samples, such as fresh crayfish meat samples and aquatic water. As represented in Table 1, no 4-NP was found in the real samples. The results found by our CD system were consistent with those that were measured using a traditional HPLC-UV method. The recovery ranging from 95.80% to 103.55% with the relative standard deviation (RSD) between 0.91% and 4.59% was achieved, indicating that the proposed method exhibited significant potential for 4-NP determination with good accuracy.

Table 1. Analytical results of 4-NP in real samples using the proposed method.

Samples	Spiked (μM)	HPLC-UV (μM)	Found (μM)	Recovery (%)	RSD (%, n = 3)
Fresh crayfish meat	0	Not detected	Not detected	-	-
	5	4.92 ± 0.17	4.79 ± 0.22	95.80	4.59
	10	10.13 ± 0.22	9.62 ± 0.27	96.20	2.81
	20	19.93 ± 0.26	20.57 ± 0.51	102.85	2.48
Aquatic water	0	Not detected	Not detected	-	-
	5	4.98 ± 0.19	5.03 ± 0.19	100.60	3.78
	10	9.89 ± 0.27	9.92 ± 0.09	99.20	0.91
	20	20.35 ± 0.48	20.71 ± 0.65	103.55	3.14

Conclusions

Herein, environment-riendly and highly fluorescent CDs were synthesized by the hydrothermal method from natural and easily available crayfish shells. The characterization techniques used indicated that the high fluorescence QY of CDs was related to the introduction of nitrogen into the carbon skeleton. The prepared CDs were employed as a specific fluorescence sensor for the facile and sensitive assay of 4-NP. The developed sensor showed a linear correlation between fluorescence quenching efficiency and 4-NP concentration from 0 to 50 μM with a LOD of 0.16 μM. Furthermore, the experimental results demonstrated that the quenching mechanism between CDs and 4-NP was attributed to static quenching. The CDs-based sensor achieved satisfactory detection recovery and low RSD when detecting 4-NP in crayfish and aquatic water samples. Therefore, the presented CDs-based sensor had a great potential application in the detection of trace 4-NP in food samples.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work reported in this article was supported by the National Key Research and Development Program of China (Project No. 2019YFC1606000), and Collaborative innovation centesr of food safety and quality control in Jiangsu Province.